1. Field of the Invention
This invention relates to a process for the transformation of Yarrowia lipolytica to improve its utilization for industrial purposes; to vectors and subclones thereof useful therefor, especially to vectors which replicate autonomously in Escherichia coli and integrate but do not replicate autonomously in Yarrowia lipolytica; to transformants of E. coli and Y. lipolytica containing said vectors and to their use for producing proteins.
2. Description of the Prior Art
Major emphasis in molecular cloning has been directed to prokaryotes, particularly E. coli, and more recently Bacillus subtilis, as host organisms. E. coli, despite its extensive use as host organism for the cloning and expression of heterologous DNA, is known to pose certain problems, such as failure to secrete proteins into its growth medium with subsequent ease of isolation thereof. The proteins thus produced intracellularly are usually not in their native state and are found as insoluble aggregates called inclusion bodies. The possible need to destroy the cells to recover the protein raises the possibility of contamination thereof with a toxic substance.
In view of the cited difficulties, B. subtilis has been turned to as an alternative host organism since it secretes protein and does not produce toxins. However, as host organism, B. subtilis is subject to certain limitations; instability of transformed strains resulting in loss of heterologous DNA, and the frequent reduction in the ability of the entering DNA to coexist with the host DNA.
In recognition of the above-mentioned difficulties with prokaryotes as host organisms, attention shifted to eukaryotes, and specifically yeasts, as host organisms. Yeasts of industrial importance, are non-toxic and can be grown to very high densities. Some species are well analyzed genetically and some species can secrete proteins.
The first transformation of a yeast, Saccharomyces cerevisiae, was reported by Hinnen et al., Proc. Natl. Acad. Sci., 75, 1929-1933 (1978) who demonstrated that a cloned segment of yeast DNA encoding the LEU2 locus could transform a nonreverting leu2.sup.- mutant of yeast to a LEU+ phenotype. This transformation was shown to result from integration into the chromosome of the plasmid containing the LEU2 segment of DNA. The integration involved recombination between homologous segments of DNA.
Hinnen et al. in "Overproduction of Microbial Products", edited by Krumphanzl et al., Academic Press, N.Y., Ch. 30, 1982, present a review of yeast transformation procedures. A prerequisite to yeast transformation was described by Ratzkin et al., Proc. Natl. Acad. Sci. 74, 487-491 (1977) in their cloning of yeast (Saccharomyces cerevisiae) LEU2 by complementation of an E. coli leuB mutation.
Sjostak et al., Plasmid 2, 536-554 (1979) describe the construction of plasmids containing the LEU2 gene of yeast and fragments of rDNA and the integration of the plasmids into the rDNA locus following yeast transformation. The basic thrust of their investigation involved the integration of a genetic marker, the LEU2 gene, inserted into the rDNA locus, as a genetic marker for mapping the rDNA. One of the reported plasmids, pSZ20, containing the Bg1II-B fragment of rDNA and the Sa1I-XhoI LEU2 fragment, is identified as a useful vector for cloning fragments of yeast DNA in yeast.
Orr-Weaver et al., Proc. Natl. Acad. Sci., 78, 6354-6358 (1981) demonstrated high frequency integration of linear plasmids derived from pBR322, all of which are nonreplicating in yeast and transform only by integration, in Saccharomyces cerevisiae when the plasmids are cut within DNA sequences homologous to the yeast chromosome.
Yarrowia lipolytica, an industrially important species of yeast, is used to produce citric acid and single cell protein. It can also be used to produce erythritol, mannitol and isopropylmalic acid. Y. lipolytica suffers from certain inherent deficiences, such as its limited spectrum of utilizable carbon sources. The overall value of Y. lipolytica could be increased by eliminating such deficiency as, for example, by introducing correcting DNA from another species. Y. lipolytica is of special interest and value because of its ability to secrete proteins (alkaline protease, acid protease and RNAse) into its growth medium, thus permitting potential recovery of heterologous proteins in the native state without the need of disrupting the producing cells.
A highly efficient system for transformation of S. cerevisiae comprising a replicating hybrid plasmid, a S. crevisiae--E. coli hybrid plasmid, which may be selected in and recovered from both E. coli and S. cerevisiae is described by Beggs, Nature 275, 104-109 (1978). High frequency transformation systems for Schizosaccharomyces pombe and Kluyveromyces lactis are reported by Beach et al., Nature 290, 140-142 (1981) and by Das et al., Current Genetics 6, 123-128 (1982), respectively.